The principal aim of the Section on Cellular Biophotonics is to use imaging techniques, such as two-photon microscopy, spectral imaging, fluorescence lifetime microscopy, and fluorescence anisotropy analysis to study how protein complexes regulate synaptic function in living cells. Recently, we have concentrated our efforts on utilizing Forster Resonance Energy Transfer (FRET) to monitor protein-protein interactions. This method has great potential for studying protein interactions because it is sensitive to changes in the distance separating two fluorophores on the 1-10 nm scale. FRET imaging in conjunction with the development of spectral variants of Green Fluorescent Protein (GFP) provides the opportunity to genetically tag synaptic proteins of interest and monitor their interactions with other labeled proteins in real time. Currently we have 3 projects in the lab. The first project is involved in building a two-photon microscope specifically designed to study protein complexes in living cells. The microscope we are assembling will be capable of simultaneously measuring time resolved fluorescence anisotropy, and the fluctuations in fluorescence intensity that can then be analyzed by fluorescence correlation spectroscopy (FCS). Our second project uses anisotropy lifetime decay analysis and FCS analysis to monitor changes in the multimeric structure of Cam kinase-II. In the brain this abundant synaptic enzyme is thought to be a calcium spike frequency detector, and has been shown to play a pivotal role in learning and memory. In the heart CaMKII activity has been linked to several forms of heart disease. Our results indicate that structural changes associated with CaM kinase-II activation can be detected using anisotropy imaging, and we now wish to image the activation of this protein complex in living zebrafish hearts, and in hippocampal spines in culture. Finally our third project is involved in revealing the function of dysferlin, a calcium binding protein responsible for two forms of muscular dystrophy. The Jain foundation has generously established a gift fund in support of this project. Dysferlin is a protein that is known to be responsible for LGMD2B/Miyoshi muscular dystrophy. Our experiments indicated that anti-sense morpholinos against Dysferlin injected into developing sea urchin embryos inhibit cell division and wound healing. Accordingly, we expanded this study to investigate the role of Dysferlin in calcium signaling. We have found that upon wounding, cells depolarize and secrete ATP by a mechanism involving agatoxin sensitive voltage-gated calcium channels. Neighboring cells respond to the secreted ATP by a mechanism thought to involve P2X receptors as well as voltage-gated calcium channels. Anti-sense morpholinos against Dysferlin block the secretion of ATP supporting the hypothesis that Dysferlin might act by mediating calcium triggered exocytosis.